Use of Time-Saving Flow Cytometry for Rapid Determination of Resistance of Human Cytomegalovirus to Ganciclovir

• Published in: Journal of Clinical Microbiology 2005-10, Vol.43 (10), p.5003-5008
• Main Authors: Lee, Gyu-Cheol, Lee, Dong-Gun, Choi, Su-Mi, Yoo, Jin-Hong, Park, Sun-Hee, Choi, Jung-Hyun, Min, Woo-Sung, Cho, Ok-Hee, Lee, Chan-Hee, Shin, Wan-Shik
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Antiviral Agents - pharmacology; Biological and medical sciences; Cytomegalovirus - drug effects; Cytomegalovirus Infections - virology; Drug Resistance, Viral - genetics; Flow Cytometry - methods; Fundamental and applied biological sciences. Psychology; Ganciclovir - pharmacology; Genotype; Humans; Infectious diseases; Medical sciences; Microbial Sensitivity Tests - methods; Microbiology; Miscellaneous; Molecular Sequence Data; Mutation; Phenotype; Phosphotransferases (Alcohol Group Acceptor) - chemistry; Phosphotransferases (Alcohol Group Acceptor) - genetics; Sequence Analysis, DNA; Time Factors; Viral Plaque Assay; Virology
• ISSN: 0095-1137; 1098-660X
• DOI: 10.1128/jcm.43.10.5003-5008.2005

There are two ways to assess the susceptibility of human cytomegalovirus (HCMV) to ganciclovir (GCV): one is a genotypic test that detects resistance-related mutations and the other is a phenotypic test that actually assesses susceptibility. The advantages of genotyping the UL97 gene are its rapidity and accuracy. However, to detect novel mutations or mutations affecting the UL54 DNA polymerase, a phenotypic test such as the plaque reduction assay (PRA) is also required. To avoid the shortcomings of PRA such as its time-consuming nature and labor-intensiveness, we developed a time-saving fluorescence-activated cell sorting (TS-FACS) technique. We obtained a GCV 50% inhibitory concentration (IC₅₀) from five clinical isolates and an HCMV laboratory strain (AD169) and compared the results with those from the PRA. The laboratory strain and three clinical isolates were sensitive to GCV. Although there was a minor discrepancy in the case of one of the three isolates, the GCV IC₅₀ values obtained by TS-FACS analysis correlated well with the results of the PRA. The remaining two isolates were resistant to GCV; one was GCV resistant due to the mutation M460V, and the GCV IC₅₀ results obtained by TS-FACS analysis and by PRA were also comparable. The advantages of TS-FACS analysis are the shorter time required, the possibility of automation, and its comparability to PRA, considered the gold standard. Thus, TS-FACS analysis may be useful as an alternative to PRA in the clinic.